A variety of molecules and/or compounds have been described for conjugating to therapeutic proteins in order to increase the half-life of the conjugated therapeutic proteins following administration to a patient (Veronese F M and Mero A, BioDrugs 2008; 22:315-29; Gregoriadis G et al., Int J Pharm 2005; 300:125-30; and Shechter Y et al.; International Journal of Peptide Research and Therapeutics 2007; Vol 13 :105-17).
Fatty acids (FA) can be conjugated to therapeutic proteins to form longer-acting derivatives. This principle for prolongation of protein or peptide half-life is based on the fact that FA can bind to human serum albumin (HSA; also referred to as albumin binding probes). The association of a FA with human serum albumin in the blood stream can lead to a substantial prolongation of the half-life of the therapeutic protein as it will recycle together with albumin through the neonatal Fc receptor. FA and derivatives thereof (e.g., corresponding methyl esters) have shown similar albumin-binding properties (Spector A A, J Lipid Res 1975; 16:165-79).
One prominent example for this longer-acting principle is insulin detemir (Levemir®) from Novo Nordisk. In insulin detemir, the carboxyl group of a FA is covalently coupled to the ε-amino group of a lysine residue of the insulin protein (see, e.g., U.S. Pat. Nos. 5,866,538; 6,011,007; and 6,869,930). Other research groups have described similar approaches (Shechter Y et al., Bioconj Chem 2005; 16:913-20; and Sasson K et al., J Control Release 2010; 142:214-20). For example, these groups describe a releasable FMOC system containing an active NHS ester for coupling to amino groups of proteins. The difference however is that in this concept the FA is linked to the protein via a functional group in ω-position thereby rendering the carboxyl group intact. Thus, prolonged-acting prodrugs can be prepared that bind to human serum albumin yet dissociate over time as the FMOC system undergoes slow hydrolysis under physiological conditions (Sasson K et al., J Control Release 2010; 142:214-20).
In addition to fatty acids, the preparation of conjugates by forming a covalent linkage between the water soluble polymer and the therapeutic protein can be carried out by a variety of chemical methods. PEGylation of polypeptide drugs protects them in circulation and improves their pharmacodynamic and pharmacokinetic profiles (Harris and Chess, Nat Rev Drug Discov. 2003; 2:214-21). The PEGylation process attaches repeating units of ethylene glycol (polyethylene glycol (PEG)) to a polypeptide drug. PEG molecules have a large hydrodynamic volume (5-10 times the size of globular proteins), are highly water soluble and hydrated, non-toxic, non-immunogenic and rapidly cleared from the body. PEGylation of molecules can lead to increased resistance of drugs to enzymatic degradation, increased half-life in vivo, reduced dosing frequency, decreased immunogenicity, increased physical and thermal stability, increased solubility, increased liquid stability and reduced aggregation. The first PEGylated drugs were approved by the FDA in the early 1990s. Since then, the FDA has approved several PEGylated drugs for oral, injectable and topical administration.
Polysialic acid (PSA), also referred to as colominic acid (CA), is a naturally occurring polysaccharide. It is a homopolymer of N-acetylneuraminic acid with α(2→8) ketosidic linkage and contains vicinal diol groups at its non-reducing end. It is negatively charged and a natural constituent of the human body. It can easily be produced from bacteria in large quantities and with pre-determined physical characteristics (U.S. Pat. No. 5,846,951). Because the bacterially-produced PSA is chemically and immunologically identical to PSA produced in the human body, bacterial PSA is non-immunogenic, even when coupled to proteins. Unlike some polymers, PSA acid is biodegradable. Covalent coupling of colominic acid to catalase and asparaginase has been shown to increase enzyme stability in the presence of proteolytic enzymes or blood plasma. Comparative studies in vivo with polysialylated and unmodified asparaginase revealed that polysialylation increased the half-life of the enzyme (Fernandes and Gregoriadis, Int J Pharm. 2001; 217:215-24).
Coupling of PEG-derivatives to peptides or proteins is reviewed by Roberts et al. (Adv Drug Deliv Rev 2002; 54:459-76). One approach for coupling water soluble polymers to therapeutic proteins is the conjugation of the polymers via the carbohydrate moieties of the protein. Vicinal hydroxyl (OH) groups of carbohydrates in proteins can be easily oxidized with sodium periodate (NaIO4) to form active aldehyde groups (Rothfus and Smith, J Biol Chem 1963; 238:1402-10; van Lenten and Ashwell, J Biol Chem 1971; 246:1889-94). Subsequently the polymer can be coupled to the aldehyde groups of the carbohydrate by use of reagents containing, for example, an active hydrazide group (Wilchek M and Bayer E A, Methods Enzymol 1987; 138:429-42). A more recent technology is the use of reagents containing aminooxy groups which react with aldehydes to form oxime linkages (WO 96/40662, WO2008/025856).
Additional examples describing conjugation of a water soluble polymer to a therapeutic protein are described in WO 06/071801 which teaches the oxidation of carbohydrate moieties in von Willebrand factor and subsequent coupling to PEG using hydrazide chemistry; US Publication No. 2009/0076237 which teaches the oxidation of rFVIII and subsequent coupling to PEG and other water soluble polymers (e.g. PSA, HES, dextran) using hydrazide chemistry; WO 2008/025856 which teaches oxidation of different coagulation factors, e.g. rFIX, FVIII and FVIIa and subsequent coupling to e.g., PEG, using aminooxy chemistry by forming an oxime linkage; and U.S. Pat. No. 5,621,039 which teaches the oxidation of FIX and subsequent coupling to PEG using hydrazide chemistry.
Notwithstanding the above materials and methods for protein conjugation, new materials and methods are desired that, for example, allow manipulation and preparation of stable protein conjugates. Although fatty acids can provide the benefit of binding HSA, fatty acids are often difficult to manipulate in an aqueous environment and can be released or removed from its protein binding partner over time.